Note: Protocols for harvesting, embedding and preparing tissue sections can be found at "Links To Protocols"

The following protocol has been optimized for staining 15 µm mouse intestine cryosections on Superfrost Plus slides (Erie Scientific Co.), using 200 µl of staining solution within a PAP pen (Polysciences, Inc.) well at room temperature. Optimal staining conditions may vary slightly depending on sample conditions and should be determined for your sample.

Dissolve the Alexa Fluor® 350 WGA (5 mg) in 5 mL PBS for a final concentration of 1 mg/mL. This can be stored at 2–6ºC if sodium azide is added at a final concentration of 2 mM. Otherwise, the solution should be aliquoted and stored at –20ºC.

Prepare a fresh stock solution of Alexa Fluor® 350 WGA in PBS at a final concentration of 0.5 mg/mL. For example, to stain 5 slides, dilute 500 µL of the stock solution with 500 µL of PBS. Centrifuge for 10 minutes at 10,000 rpm to remove any undissolved aggregates.

Flood the sections with 200 µl staining solution; protect the sample from light and incubate for 30 minutes at room temperature.

Remove the ProLong® Gold antifade reagent from the freezer and allow the vial to equilibrate to room temperature. Using an external heat source to warm the vial is not recommended, as this may decrease the long-term stability of the product.

Remove any excess liquid from the specimen and apply 1 or 2 drops (depending on the surface area of your sample) of the antifade reagent to the specimen. Cover slide-mounted specimens with a coverslip; for specimens mounted on coverslips, place a drop of antifade reagent onto a clean slide and carefully lower the coverslip onto the antifade reagent to avoid trapping any air bubbles.

Allow the mounted sample to cure on a flat surface in the dark. Curing time may vary from a couple of hours to overnight, depending on the thickness of the sample and the relative humidity of the surrounding air. For long-term storage, seal the coverslip to the slide after curing to prevent excessive shrinkage of the mounting medium, which can result in sample distortion. After sealing, store the slide upright in a covered slide box at <=–20°C. Desiccant may be added to the box to ensure that the slide remains dry.

To view the samples immediately, secure the coverslip at the corners using nail polish or hot wax to prevent the coverslip from moving. Leave the edges clear to allow the preparation to cure.

Note: The antifade properties of ProLong® Gold antifade reagent improve slightly the longer it remains in contact with the specimen. To further reduce photobleaching, minimize the exposure of fluorescently labeled specimens to light by using neutral density filters, and expose samples only when observing or recording a signal. Optimize image capture by using a minimum of optics, high–numerical aperture objectives, relatively low magnification, high quality optical filters, and high-speed film or high-efficiency detectors.